The research in Dr. Merchlinskys' laboratory is concerned with the determining the mechanism by which the poxviruses, a class of large DNA viruses, replicate their DNA. Poxviruses provide a unique system for studying the replication of DNA since the required enzymes and factors are encoded within the viral genome and DNA synthesis and processing of replicative intermediates occurs outside of the nucleus within the cytoplasm. The replication of vaccinia virus proceeds through two stages. In the first stage DNA is converted into large highly branched DNA molecules. In the second stage these structures are resolved into normal virus DNA molecules. Studies have demonstrated that a specific DNA sequence highly conserved among different poxviruses and the arrangement of this sequence is essential for processing one portion of the large DNA molecules. A second mechanism which does not use a specific DNA sequence is responsible for the resolution of the branch points in the large DNA molecules. Our effort has placed particular emphasis on discovering and understanding the DNA sequences and proteins required for the processing. This knowledge can be used for the construction of safe poxvirus vectors. The laboratory is also involved in designing new approaches to making vaccines based on poxvirus vectors and evaluating current potential orthopox vaccines. Replication of Vaccinia Virus DNA. Poxviruses provide a unique system for studying the replication of DNA. Required enzymes and factors are encoded within the viral genome and DNA synthesis and processing occurs outside of the nucleus within the cytoplasmic compartment of the cell. Therefore, it has been possible to apply genetic and biochemical approaches to the study of DNA replication. Our effort has been directed towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the replicative intermediates. This project is endeavoring to discern the cis-acting and trans-acting components required for the processing of replicative intermediates, an integral process in vaccinia DNA replication. This knowledge will be used for the construction of highly attenuated safe poxvirus vectors and for the evaluation of presently used poxvirus vectors. The replication of vaccinia virus proceeds through concatemeric highly branched intermediates that are resolved into unit length DNA molecules. Mutational analysis has demonstrated that a cis-acting DNA sequence highly conserved among poxviruses as well as the palindromic structure of the concatemer is essential for resolution of the telomere and that resolution occurs by a process involving conservative strand exchange. A model for resolution involving site-specific recombination and oriented branch migration is consistent with this data. A separate, sequence-independent mechanism is responsible for the resolution of the numerous branch points present in the replicative intermediates. Our present efforts are directed towards determining the trans-acting protein components that participate in telomeric as well as branch resolution. We have recently concentrated on the identification and characterization of a virion encoded nicking-joining enzyme. This protein is encoded at late times after infection, is included in the virion particle, and can cleave and cross-link DNA structures which mimic those found in poxvirus DNA replicative intermediates. We have identified the gene encoding for the activity by genetic complementation. Evaluation of New Generation Smallpox Vaccines. Poxviruses, and in particular vaccinia virus, have been utilized as systems for the expression of proteins in eukaryotic cells and as vectors for antigen delivery. Their ability to incorporate large amounts of DNA and wide host range leads to the expression and correct processing of a great variety of proteins in many cell lines. High level expression vectors have been constructed by expressing bacteriophage RNA polymerase genes in vaccinia. These vectors express high levels of any gene located behind the bacteriophage promoter. We have developed a series of vectors for generation of viral recombinants or conditional expression of target genes. Poxvirus vectors are customarily constructed by introducing foreign DNA into the poxvirus genome by homologous recombination. An alternative method using direct ligation vectors has been developed by our lab to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination. We have constructed and characterized direct ligation vectors engineered to contain restriction sites to fix the orientation of the insert DNA behind strongly expressing constitutive vaccinia promoters at the beginning of the thymidine kinase gene to utilize drug selection in the isolation of recombinants. These viruses provide a set of universally applicable, direct ligation, poxvirus cloning vectors, which extend the utility of poxvirus vectors for construction and expression of complex libraries. Our lab is currently working on the development of new poxvirus based vectors, including the development of direct ligation vectors for attenuated viruses with host range defects. These vectors will exhibit increased safety, especially for immunocompromised patients, since the infections will be self-limiting. One host-range, restricted-attenuated virus under consideration as a vector is modified virus ankara (MVA). In addition, the laboratory is evaluating the critical elements in the immune response generated by the present and potential attenuated vaccines against smallpox. The approaches include the elucidation of the viral proteins that elicit a humoral response, the evaluation of the heterogeneity in the vaccine and its influence on immunogenicity and the derivation of new assays to measure the critical components in the immune response to a smallpox vaccine. This project incorporates FY2002 projects 1Z01BK005009-09 and 1Z01BK005010-09.